The overall goal of this study is to determine the role of PD-1 (program death-1) and SOCS-1 (suppressor of cytokine signaling-1) in the HCV core/gC1qR-mediated IL-12 inhibition that occurs in the setting of chronic HCV infection. Our previous studies have shown that HCV core protein can inhibit IL-12 production through its interaction with a complement receptor, gC1qR, on macrophages. We have recently found that the HCV core/gC1qR-mediated T and B lymphocyte dysfunction is associated with induction of PD-1 and SOCS-1 -- two newly identified negative immunomodulators that regulate cell signaling. The role of PD-1 and SOCS-1 in the HCV core/gC1qR-mediated IL-12 inhibition in monocytes/macrophages (M/?() during HCV infection, however, remains unknown. We hypothesize that the binding of HCV core protein to gC1qR on M/?(?leads to a PD-1- and/or SOCS-1-dependent suppression of IL-12 production during chronic HCV infection. As an initial approach to test this hypothesis, we will first determine if expression of PD-1 and SOCS-1 in M/?(?is associated with loss of IL-12 during HCV infection. The mRNA expressions of IL-12 and PD-1/SOCS- 1 will be detected by real-time RT-PCR;and the levels of protein expression will be detected by Western blot or flow cytometry. We expect to see relatively lower IL-12 production in HCV patients compared to healthy subjects and that this production negatively correlates with PD-1 or SOCS-1 expression in M/?(??To analyze the mechanisms involved in the HCV core/gC1qR-mediated inhibition of IL-12 production, we will determine the physical association of gC1qR with its anchoring molecule, ?1 integrin, and with the signaling molecules PD-1, SHP-1, or SOCS-1, in M/?(?after exposure to HCV core protein using GST-pull-down and co-immunoprecipitation analysis. We will then assay for a direct role for HCV core/gC1qR-associated proteins in suppressing IL-12 production by blocking HCV core/gC1qR or PD-1/PD-L1 engagement using specific antibodies;and alternatively, by silencing SHP-1 or SOCS-1 gene expression using small interfering RNAs (siRNA). We will also analyze their potential link with Toll-like receptor (TLR) signaling pathways in M/?(?as measured by dephosphorylation of PI3K/Akt, MAPK, and JAK/STAT. We anticipate that HCV core/gC1qR engagement on M/?(?triggers the recruitment of PD-1 and SOCS-1, which delivers negative signaling to TLR-related pathways leading to IL-12 suppression and facilitating HCV persistence. The proposed studies would potentially delineate a novel mechanism by which pathogens usurp host machinery to facilitate persistence, as well as provide a rationale for designing therapeutics and vaccine strategies for chronic viral infections. PUBLIC HEALTH RELEVANCE: Chronic hepatitis C viral infection is a global epidemic with poor treatment options. How this virus leads to chronic infection remains unknown. Since gC1qR, PD-1, and SOCS-1 are inducible immunomodulators that play pivotal roles in regulating immune responses, the proposed study of their roles in suppression of IL-12 production in macrophages during HCV infection will shed new light on how this viral pathogen usurps the host machinery to facilitate persistence. An understanding of these mechanisms will provide important information for the development of effective antiviral and vaccine strategies.